Aminoacylated tRNA formation

The correctly positioned eukaryotic SOS ribosome-Met-tRNAj complex is now ready to begin the task of stepwise addition of amino acids by the in-frame translation of the mRNA. As is the case with initiation, a set of special proteins, termed elongation factors (EFs), are required to carry out this process of chain elongation. The key steps in elongation are entry of each succeeding aminoacyl-tRNA, formation of a peptide bond, and the movement, or translocation, of the ribosome one codon at a time along the mRNA. 

Four high-energy phosphate bonds per amino acid two in aminoacyl-tRNA formation, one in elongation with EF-Tu, and one in translocation from the A to the P site, involving EF-G. Forming a peptide bond requires about 5 kcal/mol. This is an expenditure of about 30 kcal/mol peptide bonds. This is the price of low entropy and high fidelity. 

Figure 38-1. Formation of aminoacyl-tRNA. A two-step reaction, involving the enzyme aminoacyl-tRNA synthetase, results in the formation of aminoacyl-tRNA. The first reaction involves the formation of an AMP-amino acid-enzyme complex. This activated amino acid is next transferred to the corresponding tRNA molecule. The AMP and enzyme are released, and the latter can be reutilized. The charging reactions have an error rate of less than 10" and so are extremely accurate.

Elongation is a cycUc process on the ribosome in which one amino acid at a time is added to the nascent peptide chain. The peptide sequence is determined by the order of the codons in the mRNA. Elongation involves several steps catalyzed by proteins called elongation factors (EFs). These steps are (1) binding of aminoacyl-tRNA to the A site, (2) peptide bond formation, and (3) translocation. 

The charging of the tRNA molecule with the aminoacyl moiety requires the hydrolysis of an ATP to an AMP, equivalent to the hydrolysis of two ATPs to two ADPs and phosphates. The entry of the aminoacyl-tRNA into the A site results in the hydrolysis of one GTP to GDP. Translocation of the newly formed pep-tidyl-tRNA in the A site into the P site by EF2 similarly results in hydrolysis of GTP to GDP and phosphate. Thus, the energy requirements for the formation of one peptide bond include the equivalent of the hydrolysis of two ATP molecules to ADP and of two GTP molecules to GDP, or the hydrolysis of four high-energy phosphate bonds. A eukaryotic ribosome can incorporate as many as six amino acids per second prokaryotic ribosomes incorporate as many as 18 per second. Thus, the process of peptide synthesis occurs with great speed and accuracy until a termination codon is reached. 

The close connection of this enzyme family with the transfer of genetic information has made it a popular object of study when dealing with questions regarding the formation and evolution of the genetic code (see Sect. 8.1). It is now agreed that the aminoacyl-tRNA synthetases are a very ancient enzyme species which do not, however, arise from one single primeval enzyme, but from at least two, corresponding to the synthetase classes. 

After formation of the initiation dipeptide, the first EF-G-dependent translocation allows binding of the third aminoacyl-tRNA in the A-site so that a tripeptide is formed. The apparent rate of this event may depend upon the nature of the initiation complex initially formed, being slower, for instance, with those containing mRNAs with an extended SD sequence than with those having either very short or no SD complementarity (C. O. G. and M. Rodnina, unpublished results). Furthermore, very powerful translocation inhibitors may block tripeptide formation to such an extent that they mimic translation initiation inhibitors. 

The formation of 0-seryl or 0-prolyl esters (Figure 1) of certain N-hydroxy arylamines has been inferred from the observations that highly reactive intermediates can be generated in vitro by incubation with ATP, serine or proline, and the corresponding aminoacyl tRNA synthetases (11,12,119). For example, activation of N-hydroxy-4-aminoquinoline-l-oxide (119,120), N-hydroxy-4-aminoazobenzene (11) and N-hydroxy-Trp-P-2 (121) to nucleic acid-bound products was demonstrated using seryl-tRNA synthetase from yeast or rat ascites hepatoma cells. More recently, hepatic cytosolic prolyl-, but not seryl-, tRNA synthetase was shown to activate N-hydroxy-Trp-P-2 (12) however, no activation was detectable for the N-hydroxy metabolites of AF, 3,2 -dimethyl-4-aminobiphenyl, or N -acetylbenzidine (122). 

Several key concepts are worth remembering. GTP is used as an energy source for translation, but ATP is used to form the aminoacyl-tRNA. The ribosome effectively has two kinds of tRNA binding sites. Only tRNAMet can bind to the P (for peptide) site, and this only occurs during the initial formation of the functional ribosome (initiation). All other aminoacyl-tRNAs enter at the A (for amino acid) binding site. After formation of the peptide bond (this doesn t require GTP hydrolysis), the tRNA with the growing peptide attached is moved (translocated) to the other site (this does require GTP hydrolysis). 

In some cases, mutation can lead to enhanced catalytic ability of the enzyme. Results for the mutation Thr-51 to Pro-51 (Wilkinson et al 1984) have been mentioned previously. The results for this and for the mutation Thr-51 to Ala-51 (Fersht e/ al., 1985) are also shown in Table 18. These mutations and that of Thr-51 to Cys-51 have been studied in some detail (Ho and Fersht, 1986). In each case it is found that the transition state is stabilized for formation of tyrosine adenylate from tyrosine and ATP within the enzyme the mutant Thr-51 to Pro-51 increases the rate coefficient for the reaction by a factor of 20. However, the enzyme-bound tyrosine adenylate is also stabilized by the mutation and this results in a reduced rate of reaction of tyrosine adenylate with tRNA (48), the second step in the process catalysed by tyrosine tRNA synthetase. Overall, therefore, the mutants are poorer catalysts for the formation of aminoacyl tRNA. The enzyme from E. coli has the residue Pro-51 whereas Thr-51 is present in the enzyme from B. stearothermophilus. The enzyme from E. coli is more active than the latter enzyme in both the formation of tyrosine adenylate and in the aminoacyla-tion of tRNA (Jones et al., 1986b). It is therefore suggested (Ho and Fersht, 1986) that the enzyme from E. coli with Pro-51 must additionally have evolved ways of stabilizing the transition state for formation of tyrosine adenylate without the concomitant stabilization of tyrosine adenylate and reduction in the rate of aminoacylation of tRNA found for the Pro-51 mutant. 

Peptide bond formation is the essential reaction catalyzed by the ribosome. Despite its importance, it was for a long time not the focus of ribosomal research, for several reasons. First, before the determination of the high-resolution ribosome crystal structures almost nothing was known about the active site. Second, under most experimental conditions accommodation of the incoming aminoacyl-tRNA is rate limiting for peptide bond... 

Figure 10 The transamidosome of Thermus thermophilus catalyzing tRNA asparaginylation. (a) Formation of the transamidosome (I) The AspRS (in blue) binds tRNA " (in gray, Kq = 2 pmol l ) before association of the amidotransferase GatCAB (in orange, Kq = 0.6 pmol l ) to form the ternary complex, (ii) The free GatCAB binds tRNA " with a poor affinity (Kd > 10 pmol r ) before association of tRNA " thus pathway (i) is preferred forthe formation of the transamidosome. (b) The catalytic cycle of the transamidosome. In the absence of free tRNA ", the transamidosome aminoacylates tRNA " with a first-order rate constant of 0.017 s (1) and amidates the tRNA "-bound Asp into Asn with a rate constant of 0.11 s (2). In the presence of an excess of free tRNA ", the first Asn-tRNA " is formed with a rate constant of 0.094 s (3), whereas the following catalytic cycles occur with a rate constant of 0.043 s (4), indicating that dissociation of the newly formed Asn-tRNA " accompanied by the disruption of the complex is rate-limiting at the steady state.

A ribozyme activity that led to RNA-modifications that are analogous to the 5 -5 pyrophosphate caps of eukaryotic RNA transcripts was selected by Huang and Yarns [84]. Actually the author s intention was to isolate ribozymes which catalyze the formation of a mixed anhydride between an amino acid carboxylate and a 5 -terminal phosphate of an RNA, an activity that is chemically analogous to the activation of amino acids by ATP catalyzed by aminoacyl tRNA synthetases. However, while the selected ribozymes did... 

All tRNA molecules have the sequence -CCA at the 3 end. This three base sequence is termed the acceptor stem. The aminoacyl-tRNA synthetases catalyze the formation of an ester between the carboxyl group of the amino acid and the 3 -OH of the ribose of the terminal adenosine moiety ... 

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